Reductive methylation of the two 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate-binding lysine residues of band 3, the human erythrocyte anion transport protein.
The bifunctional anion transport inhibitor 4,4'p-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) can react covalently with both chymotryptic peptides of band 3, the human erythrocyte anion transport protein. The functional roles of the two sites of covalent H2DIDS attachment have been investigated using reductive methylation with formaldehyde and borohydride, which is a substrate for band 3. Reductive methylation of outward facing lysing residues of intact cells can block the covalent H2DIDS reaction with both the 60,000- and the 38,000-dalton chymotryptic peptides (CH60 and CH38, respectively). The methylation does not interfere with reversible H2DIDS binding. Methylation of all copies of and 3 at both sites of covalent H2DIDS attachment inhibits Cl-Cl exchange by 75%. The same treatment does not detectably alter the apparent affinity for extracellular substrate (Br). The extent of transport inhibition by reductive methylation correlates very well with the extent of blockage of the Amino acid analysis indicates that the H2DIDS-binding lysine on CH38 rather than some coincidentally modified lysine is responsible for the inhibition. This lysine is therefore likely to be closely associated with the anion transport pathway.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D001457:Anion Exchange Protein 1, Erythrocyte; D001665:Binding Sites; D001798:Blood Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008239:Lysine; D008745:Methylation; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013267:Stilbenes
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D010446:Peptide Fragments; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid